This work was supported in part by grants from the National Institutes of Health.
1. Field of the Invention
The present invention relates to a method for site-specific cleavage of DNA at sequences not less than eight-base-pairs long, and particularly to a method comprising methylating the DNA with a sequence-specific methylase and cleaving said methylated DNA by treatment with a site-specific endonuclease enzyme.
2. Description of the Prior Art
In molecular biology, restriction endonucleases have proven to be extremely useful tools because of their ability to recognize specific sequences of DNA bases in double stranded DNAs and to cleave both DNA strands at these recognition sites. Because of this inherent ability, restriction endonucleases have provided a means for DNA mapping, sequencing and recombination in vitro. Type I restriction endonucleases recognize double-stranded DNA sequences up to 7 base pairs long but do not cleave site specifically. In contrast, type II restriction endonucleases have proved useful in molecular biology by virtue of their ability to recognize specific sequences of 4 to 6 bases in double-stranded DNA and cleave both strands at specific sites close to or in their recognition sequences (see, e.g., Roberts, R. J., Restriction and modification enzymes and their recognition sequences 11: r 135-167 (1983)).
The known recognition sites for restriction endonucleases, however, are only 4 to 7 base pairs long. Because of their short length, these sequences are likely to repeat themselves quite frequently in any one DNA strand. This phenomenon limits the selectivity activity which may be obtained in cutting specific DNA strands using restriction endonucleases by themselves.
More recently, DNA methylases were isolated. DNA methylases are site-specific enzymes which recognize a defined DNA sequence and methylate at one or more of the bases within the nucleic acid sequence (Sato, S.; Nakazawa, K.; and Shinomiya, T., "A DNA Methylase from T. thermophilus HB8", J. Biochem. 88: 737-747 (1980); Bird, A. P. and Southern, E. M., "Use of Restriction Enzymes to Study Eukaryotic DNA Methylation", J. Mol. Biol. 118: 27-47 (1978); McClelland, M., (III), "Purification and Characterization of two new Modification Methylases: M. Cla I from C. latum L and M. Taq I from T. aquaticus YTI", Nucleic Acids Res. 9(24): 6795-6804 (1981); and McClelland, M., (II) "The Effect of Site-Specific Methylation on Restriction Endonuclease Cleavage" (update), Nucleic Acid Res. 11(1): r169-r173 (1983)).
Many bacterial species have strain-specific enzymatic systems of DNA modification and restriction that serve as mechanisms for recognition and degradation of invading foreign DNAs. The essential enzymatic components of the systems are a modification enzyme that methylates the host DNA, and a restriction enzyme that recognizes as a foreigner and cleaves any DNA not carrying the host-specific methylation pattern. (McClelland, M., (I) "The Effect of Sequence-Specific DNA Methylation on Restriction Endonuclease Cleavage", Nucleic Acids Research 9(22): 5859-5866 (1981); Lacks, S.; and Greenberg, B., "Complementary Specificity of Restriction Endonucleases of D. pneumoniae with Respect to DNA Methylation", J. Mol. Biol. 114: 153-168 (1977); Geier, G. E., and Modrich, P., "Recognition Sequence of the dam Methylase of E. coli K12 and Mode of Cleavage of Dpn I Endonuclease", J. Bio. Chem. 254 (4): 1408-1413 (1979). McClelland, M., Kessler, L. G. and Bittner, M., "Site-specific Cleavage of DNA at 8- and 10-base pair Sequences", P.N.A.S. (USA) 81: 983-987 (1984), disclosing the present invention is however not considered prior art in view of its publication date.
Accordingly, there remained a need for more specific recognition sites which occur less frequently on each DNA strand, thereby affording higher selectivity in cutting DNA strands using restriction endonucleases.